Recognition of the utility of injectable suspensions of gas microparticles as useful ultrasound contrast agents for diagnostic purposes has triggered considerable research and development towards improved dispersions of gas filled microballoons or microbubbles with higher stability, better resistance to pressure variations, good echogenicity, ease of manufacture, field use and storage. Many proposals for ultrasound contrast agents with such suspensions have been made. For example, aqueous suspensions usable as imaging agents in ultrasonic echography are disclosed in WO-A-91/15244 (Schneider et. al.), WO-A-92/11873 (Beller et. al.) or EP-A-0 077 752 (Schering).
WO-A-91/15244 (Schneider et. al.) discloses microbubble suspensions containing film forming surfactants in laminar and/or lamellar form and, optionally, hydrophilic stabilizers. The suspensions are obtained by exposing the laminarized surfactants to air or a gas prior to or after admixing with an aqueous phase. Conversion of film forming surfactants into lamellar form is carried out according to various techniques including high pressure homogenisation or sonication under acoustic or ultrasonic frequencies. The reported concentration of the microbubbles in these suspensions is between 10.sup.8 and 10.sup.9 bubbles/ml. The suspensions disclosed exhibit a fairly high stability during storage.
In WO-A-94/09829 (Schneider et. al.) it is shown that concentrations of the laminar and/or lamellar phospholipids used in the preparations of very stable aqueous suspensions may be as low as to correspond to a single monomolecular layer of the phospholipid around the microbubbles in the suspension. Stable, low phospholipid content (down to a few .mu.g/ml) suspensions have been stored for prolonged periods without significant loss of microbubble count or echogenicity.
A method of imparting stability against pressure variations to suspensions of microbubbles or microballoons used as ultrasound contrast agents is disclosed in EP-A-0 554 213 (Schneider et al.). There, it has been shown that a significant enhancement of the stability of the microbubbles against collapse due to pressure variations upon injection may be achieved if commonly used air, nitrogen or other soluble gases are at least partially replaced by gases whose solubility in water expressed in liters of gas by liter of water under standard conditions divided by the square root of the molecular weight in daltons does not exceed 0.003. Gases disclosed which satisfy the above criteria are for example, SeF.sub.6, SF.sub.6, CF.sub.4, C.sub.2 F.sub.6, C.sub.2 F.sub.8, C.sub.4 F.sub.10, etc. These gases have been found to produce long lasting and in vivo very stable microballoons which in turn provide high quality echographic images.
WO-A-92/17212 and WO-A-92/17213 (Klaveness et al.) disclose ultrasound contrast agents comprising microballoons having an envelope made of non-proteinaceous crosslinked or polymerised amphiphilic substances (e.g. phospholipids) and crosslinked proteins (e.g. albumin). Microballoons are encapsulating gases such as air, oxygen, hydrogen, nitrogen, helium, argon, CH.sub.4, SF.sub.6 or gas precursors such as sodium or ammonium bicarbonate.
WO-A-93/06869 (Mallinckrodt Medical Inc.) discloses a method of ultrasound imaging of a warm blooded animal in which a pharmaceutically acceptable gas or a mixture of gases is administered to the animal and the animal is scanned with an ultrasound probe. The gases or gas mixtures are administered by inhalation as apparently upon inhalation of the mixture for a few minutes, microbubbles will form in the blood stream of a warm blooded animals and the echographic image of tissue will change. The gases and gas mixtures disclosed include oxygen, nitrous oxide, C.sub.2 H.sub.6, SF.sub.6, xenon, perfluorocarbons, etc. Useful gases and gas mixtures are those which tend to form larger bubbles in the blood and may be typified by xenon and nitrous oxide and other weakly active general anesthetics such as sulfur hexafluoride. Illustrated mixtures contain either 20% of oxygen, 60-80% of sulfur hexafluoride, and/or 20% of nitrogen, xenon, nitrous oxide or ethylene or 20% of oxygen, 20% of nitrogen and 60% of xenon or nitrous oxide. The method is based on comparison of ultrasonic signals obtained during two different scans. The first, prior to inhalation of the gas mixture and the second, some time after inhalation.
An interesting concept has been disclosed in WO-A-93/05819 (Quay). The document discloses emulsions of liquid dodecafluoropentane or decafluorobutane and sorbitol in water which upon injection form gaseous microbubbles which resist pressure variations and provide a good echogenic signal. The substances in the emulsions, although liquid at ambient temperature, are highly volatile and easily vaporize at body temperature and form gaseous dispersions in a carrier liquid containing additives and stabilisers such as sorbitol. Upon injection, the droplets of the highly volatile substance rapidly disaggregate and generate a fair amount of very persistent microbubbles. The microbubbles which only contain the chosen substance e.g. dodecafluoropentane in pure form at exclusion of air or any other gas are stabilised by stabilising agents, e.g. sorbitol, Tween.RTM. 20 and soybean oil which are present in the emulsion carrier liquid. By generalisation, Quay found that the foregoing technique was applicable to a number of other non-liquid (gaseous) chemical substances which were brought into use via a criteria defined as a relationship between volume density, solubility and diffusivity (coefficient Q). The document claims that any biocompatible gas whose coefficient Q is greater than 5 is potentially useful as an echographic agent, and a list of about 180 gases/liquids which satisfy the criteria is presented. It follows from the document that to achieve the desired properties, contrast agents are to be made with substances whose coefficient Q must be greater than 5. The criteria defined is Q=4.0.times.10.sup.-7 .times..rho./C.sub.s D where .rho. is density of the gas, D is diffusivity of the gas in solution and C.sub.s is the water solubility of the gas, and this has been developed using a simple model in which diffusivities and solubilities of gases in water are used as the approximation closest to reality. Contrast agents obtained from pure i.e. non-admixed, substances chosen according to the above criteria have shown encouraging results. Tested on experimental dogs, the contrast agents have been reported to furnish promising results in the echography of the myocardium after peripheral venous injections (see Beppu S. et al. in Proceedings from 66th Scientific Session of the American Heart Association, Atlanta, October 1993). Depending on the dose, injections of 2.2% emulsion of dodecafluoropentane have been found to provide a mean opacification during up to 85 minutes. However, with doses at which opacification of the left heart was homogeneous, there was observed a decrease in oxygen saturation of arterial blood and an increase of pulmonic arterial systolic pressure were observed.
Many of the prior art compositions have merit and many are under intensive clinical tests. Many are at various stages of development. From various reports it however appears that, to date, only a very small number of contrast agents is capable of exploiting the full range of diagnostic possibilities basically provided by ultrasound echography. Indeed, only a few contrast agents are really useful and help the medical profession to profit from the diagnostic technique which, otherwise, represents one of the best non-invasive methods for analysing organs in the human body. Not many agents allow exploitation of the full potential of the ultrasound concept and this hampers wider use of the technique and/or of the imaging agents. Experimentation with the known echographic agents has shown that some provide insufficient backscatter to ensure good intensity and contrast or provide useful images only in certain percentage of the population which limits their utility as a diagnostic tool of general use. Others, because of poor resistance to pressure variations, are too short lived to allow meaningful measurements or useful images. Typically, contrast agents whose microbubbles or microballoons are filled with gases of high solubility in water poorly resist pressure variations. Suspensions of microballoons whose envelope is made from rigid materials are also ineffective as they do not resonate sufficiently in response to the acoustic waves. Noteworthy contrast agents which have a high resistance to pressure variations are those using gases with low solubilities in the aqueous carrier. The direct consequence of low solubility is low rate of resorption and slow elimination from the body. Imaging agents made from such very insoluble gases remain in the blood circulation for prolonged periods causing relapse or recirculation of the gas microbubbles which causes interference with images produced during the initial stages of the test. Such contrast agents are generally useful for imaging the left heart but because of slow resorption or elimination, they cannot be used effectively for perfusion measurements. Perfusion measurements are usually carried out by integration of the echographic response curve, this being a typically Gaussian function, appearing after a "single pass" of the imaging agent. Relapse or recirculation after the "single pass" is therefore undesirable, as the repetition would superpose and impair the final result. It is therefore generally admitted that the persistence over a certain period of the microbubbles or microballoons endowed with high pressure resistance is more disturbing than helpful. Echographic contrast agents with very persistent microbubbles are useful only for certain studies, e.g. vascular Doppler investigations. Agents used for imaging of the left heart and myocardium should provide clear images and should have good resistance lo to pressure variation but should not be overlasting and should not disturb images created immediately upon injection. Recirculation is not a desirable feature of agents whose intended use is to cover a range of applications and clear imaging. Obviously, it is highly desirable to modulate the pressure resistance or persistence of the contrast agent after injection, i.e. to use suspensions of bubbles (or microballoons) designed with sufficient pressure resistance but with controlled life-time in the circulation. This demand is fulfilled by the invention below.